Alzheimer's Disease, β-Amyloid Protein and Tau Protein
Neurodegenerative diseases, although caused by a variety of factors, share a certain common characteristic. In effect, many of these illnesses are proteinopathies or diseases caused by misfolded, aggregating proteins (Southwell A L et al. Rev Neurosci. 21:273-287, 2010). One such degenerative disorder is Alzheimer's disease (AD), a progressive neurodegenerative disease that affects largely the elderly population leading to dementia; while by age 65 the AD prevalence in this population is 7-10%, it increases severely to about 40% in the population over 80 years of age (Sisodia S S. J Clin Invest. 104:1169-1170, 1999; Fu, H J et al. CNS Neurol Disord Drug Targets. 9:197-206, 2010).
The significance 01 this disease is highlighted by the World Health Organization's prediction that by the year 2050, AD would be the world's leading cause of death. A characteristic of AD is the deposition of the protein Aβ into clumps called oligomers on neural cells to form extracellular neuritic plaques, which seem to play a crucial role in its pathology (Crews L et al. Hum Mol Genet. 19:R12-R20, 2010; Gandy S. J Clin Invest. 115:1121-1129, 2005; Neniskyte U. et al. J Biol Chem. 286:39904-39913, 2011). In effect, the main neuropathological symptoms of AD associated with a progressive cognitive decline are the extracellular accumulation of Aβ plaques, the intracellular formation of neurofribrillary-like structures (NFTs) composed of paired helical filaments with phosphorylated tau proteins (Selkoe D J. Ann N.Y. Acad Sci. 924:17-25, 2000), and the neuronal and synaptic loss. Most Aβ consist of two isoforms, a main peptide called Aβ40 that contains amino acid residues from 1 to 40 and a minor one that is less than 5% of the Aβ, which has 2 extra amino acids, ending at residue 42 instead of 40, and which is named “long Aβ” or Aβ42 (Naslund J. et al. Proc Nat Acad Sci USA. 91:8378-8382, 1994). While both Aβ isoforms have a propensity to form β-sheets, because the long Aβ isoform, Aβ42, has more aggregability than Aβ40, it, is believed that it starts the process leading to the formation of oligomers, fibrils and plaques (Gravina S A. et al. J Biol Chem. 1270:7013-7016, 1995; Younkin S G. Ann Neurol. 37:287-288, 1995). Indeed, it has been proposed that these aggregated states of Aβ are those with the most potent neurotoxicity and responsible for AD. Support for this proposal is provided by the prion diseases, e.g. Creutzfeldt-Jakob disease, where the misfolding of amyloidogenic peptides leads to neurotoxicity, but without plaque formation (Gandy S. J Clin Invest. 115:1121-1129, 2005).
Although it is unclear if the accumulation of Aβ is either the decisive neurotoxic end-product or a by-product of an independent metabolic lesion that happens to be neurotoxic, the removal of Aβ plaques by certain immunological methods have shown a recovery of cognitive functions in a transgenic mouse model for AD (Schenk D. et al. Curr Opin Immunol. 16:599-606, 2004; Wilcock D M. et al. J Alzheimer's Dis. 15:555-569, 2008). After these results were reported using a vaccine having Aβ42 and the adjuvant QS-21, formulation AN-1792, (Schenk D. et al. Nature 400:173-177, 1999) (U.S. Pat. No. 6,905,686 B1), several groups confirmed and extended those studies concerning the effects of active immune therapy in preventing deposition of Aβ and improving the cognitive functions in a mouse transgenic model (see Tabira T. Tohoku J Exp Med. 220:95-106, 2010; Wilcock D M. et al. ASN Neuro. 3:249-258, 2011; Chackerian B. Hum Vaccin. 6:926-230, 2010; Wiessner C. et al. J Neurosci. 31:9323-9331, 2011).
While the results from a transgenic mouse model were encouraging from both the therapeutic and safety aspects, clinical studies using AN-1792 were terminated due to the meningoencephalitis triggered by the vaccine in 6% of the patients during a Phase II clinical trial. Evidently, this side effect was the result of a cell mediated inflammatory response caused by the combination of certain epitopes from the antigen Aβ42 (Gelinas D S et al. Proc Nat Acad Sci USA. 101:14657-14662, 2004) and the QS-21 adjuvant, which elicits Th1 immunity with production of cytotoxic lymphocytes or CTLs (Kensil C R et al. Dev Biol Stand. 92:41-47, 1998). In fact, autopsy of a patient immunized with AN-1792 showed the presence of T-lymphocyte meningoencephalitis and infiltration of cerebral white matter by macrophages (Nicoll J A et al. Nature Med. 9:448-452, 2003).
Nonetheless, a subsequent evaluation of the patients enrolled in that study, showed that those that developed antibodies against Aβ42 may have benefited cognitively from the vaccine (Gilman S et al. Neurology 64:1553-1562, 2005). While autopsies of some of the study participants showed a decrease in Aβ plaques, it has been difficult to establish a correlation between this decrease and improvement of cognitive functions (Holmes C. et al. Lancet 372:216-223, 2008). A more controlled study to minimize contamination by plaque Aβ has shown a strong con elation between the levels of Aβ and cognitive status (Naslund J. et al. JAMA 83:1571-1577, 2000). These results are more in line with those reported for the transgenic mouse model. An outcome of these reported studies has been a surge of activity trying to develop active and passive immunization methods targeting Aβ, in order to prevent or reduce the plaques load in the brain.
Another protein closely associated with AD is the tau protein, a microtubule associated protein that stabilizes the microtubule structure that in the adult brain is present in six isoforms, which are characterized by unusually high ratios of hydrophilic amino acids and proline. These six isoforms differ according to their content of i) tubulin binding domain repeats, i.e. 3 or 4 repeats of 31 to 32 amino acids in the C-terminal part of tau, and ii) number of specific inserts of 29 amino acids each, in the N-terminal region of the protein (Kolarova M. et al. Int J Alzheimer's Dis. 2012:732526, 2012). Tau isoforms vary in size from 352 to 442 amino acid residues. Results of this amino acid composition are its high solubility and the lack of a strict secondary structure with the peptide chain being in a random coil state (Huang H C et al. J Alzheimer's Dis, 16:15-27, 2009). Most tau isomers are found at axons, with only a few located at neuronal cytoplast and dendritic cells.
Tau's functionality is tightly regulated by its degree of phosphorylation, with too much or too little phosphorylation altering its conformation and affecting its biological functions (Ubhi K et al. Exp Neurol. 230:157-161, 2011). In pathological conditions tau is hyperphosphorylated, which results in a decrease of its binding to microtubules and the presence of isolated tau in the neurons; the isolated tau being susceptible to form paired helical filaments when 8 to 10 of the at least 30 available sites are phosphorylated. However, it has been proposed that to cause pathological changes, tau hyperphosphorylation must be at specific sites, but not others (Avila J. Front Neurosci. 4:49, 2010). Different studies indicate that in tau paired helical filaments the dominant structure is β-sheet, a structure that can contribute to conformational changes in that protein and its aggregation, which play a key role in its neuronal toxicity by forming intracellular NFTs (Kolarova M. et al. Int J Alzheimers Dis. 2012:732526, 2012). These neurodegenerative disorders caused by the tau protein's conformational alterations are known as tauopathies, with AD being the most prevalent.
A body of evidence indicates that formation of pathological tau conformers seems to occur with or after the initiation of Aβ aggregation and induction of neurotoxicity. Support for this concept is given by the fact that Aβ dimers isolated from the brains of late-onset AD patients and at subnanomolar concentrations are enough to induce hyperphosphorylation of tau at epitopes that are AD-relevant, initiating a disruption of the microtubule cytoskeleton and causing neuritic damage (Ming J et al. Proc Nat Acad Sci USA. 108:5819-5824, 2011).
However, studies with knockout and transgenic mice seem to indicate that the induction of AD may be the result of a combination of factors, which include conformational defects in Aβ, tau and perhaps some other proteins. In effect, it has been proposed that accumulation of Aβ in neurons may induce tau phosphorylation by alternative pathways, in agreement with reports showing that Aβ dimers can induce hyperphosphorylation. It has been also shown in transfected primary neurons that expression of truncated tau resulted in mitochondrial fragmentation in these cells, which was aggravated by exposure of the cells to sub-lethal concentrations of Aβ; results that indicate some kind of cooperation between those proteins (Quintanilla R A et al. Neurobiol Aging. 33(3):619. e25-35, 2012).
Therefore, it is becoming more accepted that the toxicity of Aβ is tau dependent. For instance, it has been shown that in transgenic mice expressing a truncated form of tau (Δtau) or with an absence of tau (tau−/−) and Aβ-forming, memory deficits are prevented and have an improved survival rate as compared to mice producing the normal tau protein (Ittner L M et al. Cell 142:387-397, 2010). Results that indicate that tau confers at least some of Aβ toxic properties. Thus, it now more accepted that AD may be a result of damage caused by cooperative effects between Aβ and tau; while, these 2 proteins seem to be crucial for starting AD, it is possible that other proteins may also play a role.
Similar to Aβ, because the tau protein's pathological effects seem to be the result of conformational changes, it is a suitable candidate for passive and/or active immune therapy, where the defective isoforms are removed by interactions with specific antibodies, this way reducing the extracellular Aβ neuritic plaques and intracellular tau NFTs. Indeed, it has been shown that immunization of a transgenic mouse for a tau mutant that develops early NFTs, with the peptide Tau379-408[P-Ser396,404] plus aluminum phosphate as an adjuvant, produced tau-antibodies that recognized the intracellular NFTs, reducing the aggregated tau in the brain and slowing the progression of the tangle-related behavioral phenotype (Asuni A A et al. J Neurosci. 27:9115-9129, 2007; Sigurdsson E M. J Alzheimer's Dis. 15:157-168, 2008; U.S. Pat. No. 8,012,936 B2; US 2002/0197258 A1). The fact that these tau-antibodies are attached to NFTs, shows that in addition to passing across the blood-brain barrier (BBB), they can also penetrate the cell; yet, these antibodies could not cross the BBB in wild type mice, indicating some deterioration of this barrier caused by neurodegenerative diseases. Similar results have been reported in transgenic mice expressing Tau22 and that were immunized against the pathological epitope phosphor-Ser422 combined with CFA (Troquier L. et al. Curr Alzheimer Res. 9:397-405, 2012). The results indicate that active immunization resulted in Tau clearance and the improvement of cognitive deficits caused by tau pathology.
Additional proof of the potential therapeutic efficacy of tau vaccines comes from vaccination with the peptide Tau379-408[P-Ser396,404] of transgenic mice htau/PS1, model that has an early onset and more aggressive progression of tau pathology than the htau model (Boutajangout A. et al. J Neurosci. 30:16559-16566, 2010). In this model, the results show that vaccination totally prevents severe cognitive impairment.
An explanation for the antibody-mediated clearance of tau aggregates has been obtained using an ex-vivo brain slice model. FITC labeled anti-tau antibodies show that i) the antibodies were localize on the phosphorylated tau, ii) are co-localized with markers of the endosomal/lysosomal pathway and iii) the tau-antibody complex were found together in an enriched lysosome fraction, indicating that antibody-mediated clearance of intracellular tau aggregates seems to occur via the lysosomal pathway (Krishnarnurthy P K et al. Front Psychiatry 2011; 2:59). Similar results to those obtained by vaccination have been attained by the administration of monoclonal antibodies against tau epitopes that are relevant for its pathogenic properties. Administration of antibodies against tau pathological forms to transgenic mice showing a progressive tauopathy, resulted in a reduction of tau pathology and prevention of tau intracellular buildup (Chai X et al. J Biol Chem. 286:34457-34467, 2011). Another group reported similar results using a mAb that targets the pathological phospho-tau protein, a treatment that resulted in decrease of both tau pathology and functional impairments in a transgenic mouse model (Boutajangout A. et al. J Neurochem. 118:658-667, 2011).
Although the immunotherapy approach for treatment of tauopathies has been focused on the hyperphosphorylated tau, some groups have proposed that tau oligomers, i.e. aggregates between the size of monomers and NFTs, should be the target of immunotherapy. The bases for this proposition is the evidence that neuronal loss precedes NFTs formation and that tau oligomers can cause neurodegeneration and memory impairment in the absence of Aβ (Kayed R. Hum Vaccin. 6:931-935, 2010). Yet, that immunization with the full-length tau resulted in a severe autoimmune reaction, mules out the use of soluble tau as an immunogen and stresses the need for identification of epitopes highly specific for early tau damage. Hence, the available data shows that immunotherapy producing antibodies that target different tau protein structures is a viable option in AD treatment, possible in conjunction with immunotherapy of Aβ because of the apparent synergism between Aβ and tau pathological effects.
The Immune System
Vertebrates exhibit both non-specific immunity (also referred to as innate immunity) and specific immunity (also referred to as acquired or adaptive immunity). Innate immunity ligands, which are pathogens' products, upon recognition by receptors encoded in the animal's genome trigger a defensive response involving cytokine production, activation of complement and natural killer cells and the identification and removal of foreign substances by specialized white blood cells; i.e. these ligands are exogenous adjuvants or immune agonists (Akira S et al. Cell 124:793-801, 2006).
Innate immunity may also start the specific humoral and cell-mediated immunities, carried out by B and T lymphocytes together with other cells. B cells participate in humoral immunity or Th2 immunity when activated to produce antibodies by antigen presenting cells (APCs) and CD4+Th2 helper T cells (Iwasaki A et al. Science 327:291-295). The Th2 immunity is characterized by the production of non-cytolytic antibodies and anti-inflammatory cytokines. Cell-mediated immunity or Th1 immunity, involves cytokine production by Th1 helper T cells, activation of macrophages and antigen-specific CD8+ cytotoxic T-cells (CTLs) to destroy pathogens. Activation of CD4+ and CD8+ T cells requires two signals: one derived from the T cell receptor (TCR) interaction with antigen-major histocompatability (MHC) complexes on APCs, and the second a co-stimulatory signal delivered by CD80 or CD86 ligands (also known as B7-1 and, B7-2, respectively) on the APCs when binding to the CD28 surface receptor on T cells (Smith Garvin J et al. Annu Rev Immunol. 27:591-619, 2009). The result of a concerted stimulation with CD80 is Th1 immunity with the production of pro-inflammatory cytokines and CTLs that are crucial to destroy tumor and virally infected cells (Feili-Hariri M et al. J Leukoc Biol. 78:656-664, 2005).
Immunization with the Aβ42/QS-21 vaccine formulation of transgenic mice and patients in the Phase 1 clinical studies elicited a Th2 immune response, while the patients in the Phase 2 clinical studies exhibited a Th1 immune response. However, the vaccine formulation used in the Phase 2 clinical studies that elicited Th1 immunity was not identical to the one used either in the mouse model or the Phase 1 clinical studies, which elicit Th2 immunity. The difference between both formulations was that the one that elicited Th1 immunity contained a non-ionic detergent, polysorbate-80, presumably to aid the manufacturing process and stability of Aβ42; in contrast, the formulation that elicited a Th2 immunity was lacking the detergent (Schenk D. et al. Curr Opin Immunol. 16:599-606, 2004).
Thus, as a result of the active immune therapy results, a great deal of activity has been devoted to design new peptide antigens related to Aβ and denominated here “Aβ-derived peptides” to maximize the production of anti-Aβ antibodies, while restricting the Th1 immune response. For instance, shorter Aβ-derived peptides lacking T-cell epitopes have been used in vaccines that in the transgenic mouse model produced antibodies that either pass across the BBB and reduced the levels of insoluble Aβ or act as a peripheral sink by clearance of circulating Aβ (Petrushina I. et al. J Neurosci. 27:12721-12731, 2007; Lemere C A et al. Curr Alzheimer Res. 4:427-436, 2007; Lemere C A. Prog Brain Res. 175:83-93, 2009; Verdoliva A et al. Hum Vaccin. 6:963-947, 2010, Fu H J et al. CNS Neurol Disord Drug Targets, 9:197-206, 2010).
A different tactic to induce only a Th2 immune response has been the use of mutated Aβ (Cao C. et al. BMC Neurosci. 9:25, 2008) and of cyclic peptides from Aβ (Hoogerhout P. et al. PLoS One. 6(4):e19110, 2011). These and other Aβ-derived peptides have been described in the following patents and patent application U.S. Pat. No. 6,787,637; U.S. Pat. No. 6,861,057; U.S. Pat. No. 7,067,133 B2; U.S. Pat. No. 7,588,766; U.S. Pat. No. 8,022,180 B2; U.S. Pat. No. 8,034,348 B2; U.S. Pat. No. 8,034,353 B2; US 2002/0094335 A1; EP 1420815 B1; 2007/0135337 A2; US 2009/0202627 A1; US 2010/0062011 A1; US 2011/0002949 A1; US 2011/0182928 A1; US 2011/0206706 A1; US 2011/0206742; US 2011/0262458 A1; US 2012/0052086 A1; 2012/0315321 A1). Other methods to elicit Th2 immunity against Aβ involve the use of DNA vaccines to express antigens similar to those indicated above (EP 2173375 A1; US 2012/0014987 A1), alone or in conjunction with a virus or a virus like particle (U.S. Pat. No. 6,719,970; U.S. Pat. No. 6,964,769; U.S. Pat. No. 7,264,810; U.S. Pat. No. 7,279,165; U.S. Pat. No. 7,479,280; U.S. Pat. No. 7,875,450; U.S. Pat. No. 7,8,318,687).
Similar to Aβ-based vaccines, tau-based vaccines apparently have the same restrictions about Th1 immunity. Administration to a transgenic mouse model for AD of lipopolysaccharide (LPS), a TLR4 ligand and known inducer of inflammation, resulted in activation of microglia and tau hyperphosphorylation, but it did not affect Aβ (Kitazawa M et al. J NeuroSci. 25:8843-8853, 2005). LPS exacerbated tau pathology via the kinase cdk5. Thus, it is apparent that any immune response in the CNS must be confined to Th2 immunity producing antibodies only. Another report showing the damaging effects of an inflammatory response on the CNS, is one in which full length tau was administered with CFA, a powerful Th1 inflammatory adjuvant. Vaccinated non-transgenic mice developed autoimmunity and showed the histopathologic features typical of AD and tauopathies, stressing the dangers of using full length tau with presumably T and B epitopes and a Th1 adjuvant (Rosenmann H et al. Arch Neurol. 63:1459-1467, 2006). Because of these results, it is obvious that tau-based vaccines must be formulated with only tau's B epitopes and without a Th1 adjuvant, like lipid A or CpG, to avoid stimulation of a damaging Th1 inflammatory response; situation that parallels that of the Aβ-based vaccines for the treatment or prevention of AD and that should be the norm for any vaccine that stimulates an immune response that acts on the CNS.
Due to the side-effects caused by a pro-inflammatory Th1 immune response, such as that elicited by QS-21 or lipid A, alternatives to stimulate only Th2 immunity to produce protective antibodies have been investigated, including other adjuvants, substitute administration modes, some delivery systems and different carriers.
One approach has been the use of Aβ40 without any adjuvant and administered intra-nasally, to produce antibodies against the amino acid sequence 1-15, recognized as the B-cell epitope. This immunization mode resulted in a Th2 immune response and reduced levels of Aβ plaques in transgenic mice (Town T. CNS Neurol Disord Drug Targets. 8:114-127, 2009).
Another group has used mutated Aβ peptides, but without an adjuvant to stimulate in mice Th2 immunity with antibody production, apparently with good results. Another method to avoid the inflammatory response has been the use of adjuvants that stimulate Th2 immunity with antibodies production. For instance, immunization of younger transgenic mice with Aβ42 without T-cell epitopes plus alum yielded an effective antibody response with reduction of Aβ burden but no cerebral microhemorrhages. Yet, that immune response did not occur in older mice, a deficient response that may be due to immunosenescence as well as the weak adjuvant used (Asuni A A et al. Eur J Neurosci. 24:2530-2542, 20061, a critical factor considering that AD affects predominantly the ageing population.
To boost the Th2 immune response stimulated, by alum, the switching of alum to Quil A, a preparation of quillaja saponins that contains QS-21, has been tried (Ghochikyan A et al. Vaccine 24:2275-2282, 2006). According to these authors, changing from alum to Quil A increases the antibodies levels without changing the Th2 antibodies profile; yet, there is a significant reduction of the IgG1/IgG2a ratio, a strong indicator of a Th1 biased immunity. While the discrepancy about the antibodies' profile can be explained by the fact that Quil A and QS-21, stimulate both Th1 and Th2 immunity, a reduced IgG1/IgG2a ratio after treatment with Quil A, shows that IFN-γ, a Th1 cytokine, is being secreted (Finkelman F D et al. J Immunol. 140:1022-1027, 1988). Also, the large increase in IgG2b reported after the switch to Quil A, indicates secretion of IL-12, a Th1 driving cytokine (Germann T et al. Eur J Immunol. 25:823-829, 1995).
Although it has been presumed that a switch from alum to Quil A does not induce Th1 immunity while enhancing the production of anti-Aβ antibodies, and thus proposed as a safe and beneficial treatment for AD patients (see Fu H J et al. CNS Neurol Disord Drug Targets, 9:197-206, 2010), it is possible that the change in immunity triggered by Quil A and leading to stimulation of Th1 immunity and down-regulation of Th2 immunity, may lead to undesirable autoimmune responses against the naturally occurring A. Other strategies involve the use of Aβ peptides that are B cell, epitopes and that are connected sequentially either to T cell epitope sequences unrelated to Aβ or are conjugated to a carrier protein that would, then stimulate the T cells, this way avoiding an anti-Aβ inflammatory response (Agadjanyan M G et al. J Immunol. 174:1580-1586, 2005; Verdoliva A et al. Hum Vaccin. 6:936-947, 2010). Conjugates of the polysaccharide mannan, a Th2 adjuvant, and an Aβ-derived peptide, Aβ28, have been used to immunize APP transgenic mice; while the immunization produced antibodies that prevented Aβ-plaque deposition, it also increased the risk of microhemorrhages in the brains of vaccinated animals; apparently a result of the anti-Aβ antibodies stimulated by the mannan conjugate (Petrushina I et al. J Neuroinflammation 5:42, 2008) Several of these antigen constructs are the subject of issued patents or pending patent applications.
To prevent Th1 immunity while inducing an effective Th2 immune response with protective antibodies, Aβ-derived peptides that are B-cell epitopes have been expressed or covalently conjugated to virus like particles (VLP) derived from the E. coli phage Q P, which provides a scaffold to link the peptides as well as T-helper cell epitopes (Wiessner C et al. J Neurosci. 31:9323-9331, 2011; Chackeran B et al, Vaccine 24:6321-6331, 2006; U.S. Pat. No. 6,719,978 B2; U.S. Pat. No. 6,964,769; U.S. Pat. No. 7,264,810; U.S. Pat. No. 7,279,165; U.S. Pat. No. 7,320,793; U.S. Pat. No. 7,371,572 B2; U.S. Pat. No. 7,479,280 B2; U.S. Pat. No. 7,494,656; U.S. Pat. No. 7,875,450 B2; US 2004/0141984 A1; US 2009/0246215 A1; US 2013/0011431 A1). The use of VLPs apparently facilitates the production of antibodies against tolerogens or self-antigens, such as Aβ.
An alternative strategy has been the use of retrovirus-like particles having only the gag and pol proteins from murine leukemia virus, but displaying Aβ-peptides, i.e. Aβ retroparticles, to stimulate Th2 immunity against Aβ (Back P et al. J Immunol. 182:7613-7624, 2009).
In all cases, the vaccines have been designed to stimulate production of Th2 immunity with anti-Aβ antibodies that reduce the Aβ load, while avoiding Th1 immune pro-inflammatory responses, either without adjuvants or with adjuvants that elicit only Th2 immunity. Hence, most of the attention has been focused on the antigen rather than the adjuvant component; evidently a result of the inflammatory response observed during the phase 2 clinical studies with the Aβ vaccine AN-1792 containing QS-21. Of significance is that the Th1 inflammatory response took place only after modification of the original vaccine formulation by adding the non-ionic detergent polysorbate-80, for use in the phase 2 clinical studies; a response that has been explained by the exposure of Aβ T-cell epitopes due to the detergent combined with the use of an effective Th1 adjuvant. However, it is most likely that the main target of the detergent action was the QS-21 adjuvant rather than the antigen, i.e. it has been shown that addition of non-ionic detergents to QS-21, Quil A and other q. saponin analogs, causes a large enhancement of their adjuvanticity, 10-fold or more based on the IgG titers elicited in the absence and presence of polysorbate-80 (EP1009429 A1).
Thus, it is feasible that an incipient immune response, result of a supposedly suboptimal formulation, was magnified by the large increase in the QS-21 adjuvant activity. It is dubious that low concentrations of the non-ionic detergent polysorbate-80, would have significant effects on the Aβ structure; i.e. oligomeric Aβ can only be dissociated by using high concentrations of formic acid, strong chaotropic agents, like Gu.HCl, or detergents as sodium dodecyl sulfate (SDS) under stringent conditions (Masters C L et al. Cold Spring Harb Perspect Med. 2:a006262, 2012). However the use of Aβ-derived peptides lacking T-cell epitopes has confirmed that those epitopes, in combination with what it was apparently a “high dose” of QS-21, were responsible for the Th1 pro-inflammatory response.
Analogous to the Aβ vaccines constrains on stimulation of Th1 immunity, the tau vaccines that have shown beneficial results in transgenic mouse models for AD, are those with phosphorylated short tau peptides as immunogens, in some cases conjugated to carrier proteins. In contrast, tau vaccine formulations containing the full length tau plus a Th1 adjuvant, like LPS or CFA, stimulated a damaging inflammatory response. Yet, the alternative of using Aβ or tau-based vaccines without an effective adjuvant or one like alum, a Th2 adjuvant that stimulates IgG production but fails to stimulate an effective immune response in aged mice, presents a serious problem because in humans and some mammalians, the target populations for AD or AD-like conditions are the aged ones. This situation stresses the need for effective adjuvants in the AD vaccines to stimulate a beneficial Th2 immune response in the elderly while overcoming the effect of immune senescence and preventing tolerance.
Immune senescence or the deter-oration of the immune system linked to aging, is one of the main challenges to the development of vaccines for the elderly (Gruver A et al. J Pathol. 211:144-156, 2007; Ron-Harel N et al. Trends Neurosci. 32:367-375, 2009); Ongradi J et al. Immun Ageing 7:7, 2010). Aging affects APCs, i.e. dendritic cells and macrophages, by reducing the expression of toll-like-receptors (TLRs) associated with innate immunity (Aspinall R et al. Immunity and Ageing 4:9, 2007), and the co-stimulatory ligands CD80 and CD86 associated with adaptive immunity.
Another significant aging-related change is the decrease in expression of the CD28 receptor for the B7 ligands in CD8+ and CD4+ T cells (Plackett T P et al. J Leukoc Biol. 76:291-299, 2004); these reduced expressions of the APCs' co-stimulatory ligands and T cells' CD28 receptor, are major factors contributing to the immunity decline in the elderly, which leads to tolerance. Immune senescence also occurs in the brain, causing the microglia, which are the macrophages equivalent (Gemechu J M et al. Front Cell Neurosci. 6:38. 2012; Wrona D. J Neuroimmunol. 172:38-28, 2006; Streit W J et al. Aging Dis. 1:254-261, 2010), to function atypically and promote neurodegeneration (Luo X-G et al. Mol Neurodegener. 5:12, 2010). However, as the BBB acts as a filter that shields the brain from molecules and cells from the blood milieu, the brain is an immune privileged organ; thus, it is unlikely that Aβ or tau-derived antigens from vaccines would be processed by the brain's microglia. Hence, like other antigens, initial processing of tau or Aβ-derived antigens should take place at the APCs, with the subsequent secretion of Th1 or Th2 cytokines and interactions with T cells leading to T cell activation and production of CD4+ or CD8+ T cells, functions that can be modulated by different adjuvants (Marciani D J. Drug Discov Today. 8:934-943, 2003). While there is evidence that in AD the peripheral T cells, CD4+ and CD8+ T cells, can enter the brain and infiltrate areas with Aβ-plaques, T cells may also exert, their effects without entering the brain, via Th1 and/or Th2 cytokines, e.g. IFN-γ, TNF-α, IL-10, and other factors (Jozwik A et al. PLoS One 7(3):e33276, 2012; Fisher Y et al. PLoS One 5(5):e10830, 2010).
Thus, immune senescence may affect the immune system by blocking the production of cytokines by anergic peripheral T cells and imposing tolerance; a situation that may impact the production of protective antibodies. In fact, down regulation of the co-stimulatory APC ligands, CD80 and CD86, and/or their T cell's receptor CD28, by eliminating the required co-stimulatory signal causes T cell anergy (Mondino A et al. J Leukoc Biol. 53:805-815, 1994; Su-Yi T et al. Current Opin Cell Biol. 14:575-580, 2002). Because this condition may be common in the elderly AD population, it is unlikely that vaccines without an effective adjuvant or immune agonist would stimulate a useful protective antibody response against Aβ or tau protein in many of those patients. Essentially, the adjuvant(s) required for an AD vaccine would need to deliver an alternative T cell co-stimulatory signal to replace the one delivered by the down-regulated CD80/86 ligands to allow T cell activation and elicit only a Th2 immune response with production of protective antibodies, a task that adjuvants like IFA, alum and many others cannot deliver.
Saponin Adjuvants
The only well-characterized exogenous immune agonists or adjuvants that can deliver an alternative co-stimulatory signal are Quil A and its components, such as QS-21, QS-18, QS-17 and QS-7. Structurally, these quillaja saponins are glycosides that have as an aglycone a lipophilic triterpene, which is linked to two oligosaccharide chains bound at positions C-3 and C-28. Because of the presence of both hydrophilic and lipophilic structures, saponins have an amphipathic character with detergent-like properties. It is due to these properties and that the triterpene can insert into the cholesterol of the membrane's lipid bilayer, that quillaja saponins have a translocating capacity, i.e. allow the passage of proteins across the cellular membranes, such as the cell and endosomal membranes, directly into the cytosol.
Most of these saponins, with the exception of QS-7 that is acetylated, have their fucosyl residue acylated with an acyl-acyloyl moiety composed of two 3,5-dihydroxy-6-methyloctanoic acid residues linked in tandem (R Higuchi et al. Phytochemistry 26:229-235, 1987; ibid, 27:1165-1168, 1988) and de-acylation occurs under mild conditions, i.e. above pH 6 and at room temperature (RT), a situation that apparently results in a loss of these saponins' capacity to insert and pass across the cell's membrane, to deliver an antigen directly into the APC's cytosol to trigger CTL production. Structure/function studies have shown that the aldehyde group is essential to stimulate immunity and that its modification results in a loss of the immune stimulatory capacity (Soltysik S et al. Vaccine 13:1403-1410, 1995). In effect, synthetic mannosylated triterpene glycosides based on oleanolic and glycyrrizhic acids that lack an aldehyde group, do not show immunological activity (Dairies A M et al. Bioorg Med Chem. 17:5207-5218, 2009), a result of the lack in those glycosides of a group capable of providing a co-stimulatory signal to the T cells. In fact, from a variety of triterpene saponins, only those carrying an aldehyde group, i.e. Quillaja saponins, >Gypsophila saponins and Saponaria saponins, have adjuvant activity (Bomford R et al. Vaccine 10:572-577, 1992). Furthermore, it is evident that the lipophilic acyl moiety of quillaja saponins is also required for stimulation of Th1 immunity with CTL production, as de-acylated quillaja saponins or DS-QS (FIG. 1) stimulate Th2 immunity without CTLs (Marciani D J et al. Int. Immunopharmacol. 2001; 1:813-818; Liu G et al. Vaccine 20:2808-2815, 2002).
Hence, it is apparent that an acyclic alkyl chain, bound to the triterpene is essential to allow its passage, together with the antigen, across the cell and endosomal membranes for processing by the endogenous pathway, a requirement to yield Th1 immunity with CTL production (Marciani D J et al. Vaccine 21:3961-3971, 2003). Of relevance is that while a small co-stimulatory Schiff-base-forming drug, 4-(2-formyl-3-hydroxyphenoxymethyl benzoic acid) or tucaresol (Rhodes J et al. Nature 377:71-75, 1995; U.S. Pat. No. 5,958,980), favors a Th1-biased immunity with a Th1-type cytokine profile and concomitant CTL production, DS-QS and other non-acylated aldehyde carrying saponins stimulate Th2 immunity. Important for an AD vaccine is that while DS-QS stimulates a Th2 immunity, because of its aldehyde group still has the capacity to deliver to T cells the co-stimulatory signal needed for their activation and prevent anergy (Hall S R et al. Immunology 104:50-57, 2001).
Moreover, the Th2 immune response seems to be independent of the presence or absence of T cell epitopes in the antigen, i.e. while mice immunized with, ovalbumin (OVA) plus QS-21 or Quil A developed a Th1 immune response with the concomitant production of OVA-specific CTLs, mice immunized with OVA and DS-QS develop a Th2 immune response without either CTL production (Marciani D J et al. Int. Immunopharmacol. 2001; 1:813-818) or secretion of IFN-γ. Though OVA has a peptide sequence, OVA323-339, which has multiple overlapping T cell epitopes and MHC-binding registers (Robertson J M et al. J Immunol. 164:4607-4712, 2000), immunization with DS-QS failed to elicit Th1 immunity and CTL production; in contrast, immunization with the acylated glycosides Quil A or QS-21 plus OVA, stimulated both a strong Th1 immunity and OVA-specific CTL production.
These results show that DS-QS and similar natural or synthetic non-acylated saponins modulate the immune response against OVA in a manner independent of the presence of T cell epitopes in the antigen. In fact, saponins that have small acyl groups like acetyls, e.g. QS-7 (U.S. Pat. No. 6,231,859 B1) (FIG. 2), behave to some degree similar to DS-QS. However, QS-7 at higher doses stimulates the production of antigen-specific CTLs (U.S. Pat. Nos. 6,231,859 B1; 6,524,584 B2), indicating that some degree of acylation is required for these glycosides to stimulate Th1 immunity.